Limulus Amebocyte Lysate (LAL) assays

The key methodological principle of chromogenic assays is to reveal the presence of the analyte in a test sample via chemically-induced visible color changes. The resulting color is then measured using spectrophotometric methods to reveal the concentration of the analyte in the sample.

This method forms the basis of the chromogenic LAL assay. The LAL reagent is mixed with a chromogenic reagent (a peptide connected to p-nitroaniline, a yellow colorant) to produce a synthetic chromogenic substrate. This is added to the test sample to create the test solution, which is then incubated.

If endotoxins are present in the sample, the subsequent enzymatic reactions of the LAL reagent break the peptide bonds connected to the p-nitroaniline molecules, releasing the yellow color into the solution. The more endotoxin present, the more yellow the solution will become. This can be quantitated using an absorbance plate reader to reveal the specific endotoxin concentration. 

Assay principle

When endotoxin binds to Factor C, Factor B is activated that cleaves the proclotting enzyme into its active form. The resulting clotting enzyme will cleave the chromogenic substrate, releasing the yellow color that can be measured at 405-410 nm with an incubating absorbance reader.

Kinetic-QCL® Kinetic Chromogenic LAL Assay kits

Our Kinetic-QCL® Kinetic Chromogenic LAL Assay kits have a sensitivity range from 0.005 to 50 EU/ml. These kits contain co-lyophilized lysate/substrate and matched control standard endotoxin. Bulk kit configurations are also available. Kinetic chromogenic lysate and matched control standard endotoxin are packaged separately but should be ordered together. The chromogenic nature of the assay makes it the most appropriate choice for testing small volume parenterals, vaccines, antibiotics and biologicals. It is also ideally suited to testing inhibitory products, which may interfere with the clotting mechanism in turbidimetric and gel clot LAL assays.

eLearning module: Working with photometric assays

This module covers the basic principles of working with photometric methods, includes a demonstration assay video and sections on calculating the MVD, product characterization, product validation, and routine testing.

Turbidimetry measures the presence of solid particles in an otherwise homogeneous solution (the turbidity). The general procedure involves measuring the wavelength of light passing through the solution using spectrophotometric methods. This reveals the concentration of the substance and the substances present in the solution that are causing the turbidity.

In turbidimetric LAL tests, the LAL reagent is added to the test sample to make a solution. If endotoxins are present in the sample, then the clotting reaction of the LAL reagent produces a solid mass (i.e., the clot or gel) in the solution. The resulting degree of turbidity is subsequently measured to determine the presence and amount of endotoxin.

Assay principle

The enzymatic cascade for the kinetic turbidimetric assay is nearly identical to that of the gel clot assay. The cascade ultimately results in the formation of coagulin which is detected as the appearance of turbidity.

The gel clot LAL assay is the simplest form of the LAL assay and can easily be carried out in most laboratories. It provides an easy to read qualitative result indicating the presence or absence of endotoxin in the test sample. It is most often mentioned as the official referee test in pharmacopeial monographs.

The gel clot LAL assay involves mixing the LAL reagent with the test sample in a tube, which is then incubated. If endotoxin is present in the sample, the clotting response by the LAL reagent will be initiated. After the incubation period, the tube is examined by eye to determine whether a clot (or gel) has formed at the bottom of the tube. A positive result is indicated by the presence of a clot which stays at the bottom of the tube upon gentle inversion, whereas a negative result is indicated by liquid flowing down the inside of the tube.

Assay principle

The first stage of the gel clot LAL assay is the binding of endotoxin by a key protein, known as Factor C. Factor C is an inactive protease that becomes activated once bound to endotoxin. Activated Factor C, then acts on another inactive protease, Factor B. Activated Factor B in turn activates a pro-clotting enzyme. The proclotting enzyme becomes the clotting enzyme. Coagulogen in the presence of the clotting enzyme becomes coagulin – which forms the clot.